CHARACTERIZATION OF STREPTOMYCES GRISEUS BACTERIOPHAGES1RENEE R. ALEXANDER' AND ELIZABETH McCOY

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin

Received for publication March 8, 1956

Because the Streptomyces grisew bacterio-phages have been of industrial concern, a numberof the viruses active against this host have beengathered. The Wisconsin collection to be re-ported here comprises seven phages of which twoare of industrial origin, four isolated at Wisconsin(Hoehn, 1949), and one recovered from a lyso-genic host strain. Because of the variabilityfound in their identification by lytic patterns, aneed for a stable classification arose. It was alsoof interest to apply to these phages, whose hostis an actinomycete and thus of mycelial nature,some of the criteria for classification of phages ofthe Eubacteriales.Burnet (1933a and b) showed that serological

properties offer probably the most significantdifferentiating character for bacteriophage classi-fication and Delbrilck (1946) pointed out thatthe serological classification often correlates withthe morphology of the viruses as shown byelectronmicrographs. On this basis, various host-virus systems have since been characterized, suchas the "T" system of phages active againstEscherichia coli strain B (Delbriick, 1946, andAdams, 1952), the "M" system viruses whichattack Bacillus megaterium (Friedman andCowles, 1953), the viruses active on Micrococcuspyogenes var. aureus (Rountree, 1949), and thoseon Streptococcus lactis (Wilkowske et al., 1954a).In addition to serology, biological propertiesincluding growth curves of the phages on theirhomologous hosts, and physical inactivationcharacteristics such as heat resistance and pHsensitivity are included in most classificationsystems so far proposed. The present investiga-tion was directed along similar lines in order tocontribute an additional phage system to aid inthe taxonomy of bacterial viruses in general.

1 Published with the approval of the Directorof the Wisconsin Agricultural Experiment Station.This work was supported in part by grants fromCommercial Solvents Corporation, Terra Haute,Ind., and Bristol Laboratories, Syracuse, N. Y.

2 Present address: Laboratory of Bacteriology,Cornell University, Ithaca, N. Y.

MATERIALS AND METHODS

The collection consists of seven phages forwhich four strains of Streptomyces griseus serve ashomologous hosts. The phages with their hostsof isolation are: W-1 and W-3 on S. griseus 1945;W-1A, W-2a, and B on strain 1947; W-5 onstrain 1949; and phage C-131 on the C-131strain. Of these W-1, W-2a, W-3 and W-5 wereisolated at Wisconsin whereas B and C-131 werereceived from industrial concerns. Phage W-1Awas derived from a suspension of W-1 phage andhas been identified as being carried by thelysogenic S. griseus 1945, the host of W-1.The phages were propagated in 500 ml flasks

with 100 ml of medium containing in g per L:yeast extract 3, glucose 2.5, peptone 1 and tapwater 1000. Two to three ml of a heavy suspen-sion of spores of the host were added and theflasks incubated on a rotary shaker (350 rpm) at30 C for 6-12 hr. At this age the spores were wellgerminated and a young mycelium susceptible tophage lysis had developed. This culture was theninoculated with 1 ml of the virus at a concentra-tion of 107 to 108 particles per ml. The flask wasreturned to the incubator for 2-4 hr, then re-moved from the shaker to stop growth of thesubmerged mycelium and allowed to stand undis-turbed for another 6 to 8 hr. In such a 24 hr cycletiters of about 109 phage particles per ml wereregularly obtained. At time of harvest, the celldebris was eliminated by centrifugation and thesupernatant containing the virus was passedthrough a sterile Seitz filter, bottled and storedat 4 C where it remains stable for several months,the pH being in the range of 6.8 to 7.2. Thephages were titered on assay medium of thefollowing composition in g per L: glucose 5, yeastextract 10, K2HPO4 1, agar 15, tap water 1000.The plates were slightly dried by pouring oneday in advance of use. Serial tenfold dilutions ofthe phage suspension were made in 0.1 per centpeptone water (preferred over distilled water forits greater protective effect on the phage). Onetenth ml of diluted virus was transferred onto the

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agar surface and to this was added 0.1 ml of aspore suspension of the proper host; the mixturewas then spread evenly over the surface of theplate. Following 24-36 hr incubation, the timebeing determined by the rate of growth of thehost strain, those plates representing the highestdilution at which 30-300 plaques developed werecounted and the proper titer calculated. Thisspreading technique is used as it provides con-ditions for rapid development of aerial myceliumin the growth of the Streptomyces species (Chang,1953).The methods employed for the general classifi-

cation of S. griseus phages were adapted fromthose of Adams (1950). For the production ofantiserum, a total of 10 ml of virus at a concen-tration of 101-109 particles per ml was injectedper rabbit over a period of 25 days. The neutral-izing power of an antiserum was determined interms of a constant, k. The serum was diluted to1:100 and 1:1000 and 1 ml of the antigen con-tamiing 107 phage particles per ml was added to9 ml of each dilution of antiserum at 37 C. At 5min intervals 0.1 ml samples of the phage-serummixture were withdrawn and added to 9.9 iial ofdiluent to stop the neutralizing action. Fromthese tubes, further appropriate dilutions weremade and samples plated by the regular assayprocedure. The percentage of phage neutralizedwas then determined and a velocity constant kcalculated at 99 per cent neutralization: k = 2.3D/t X log po/p; where D is the dilution of anti-serum used in the determination; t, the timerequired to obtain 99 per cent neutralization; po,the plaque count before the addition of antiserumand p, the plaque count after neutralization.Antisera were then tested against their homolo-gous phages and against all other phages in theseries to detect cross-neutralization reactions.For one-step growth experiments the procedure

of Adams (1950) was followed in principle in thedeterminations of latent periods and burst sizes.In these experiments young germinated spores ofS. griseus, 5 to 6 hr old, were used at a concentra-tion of 5 X 107 cells per ml and after addition ofphage, an adsorption period of 30 min wasallowed, since this is feasible with the long latentperiods of this phage system.

For all physical inactivation experiments thestock phages were adjusted to a concentration of107 particles per ml. The inactivating agent wasapplied to 1 ml of this phage diluted in 9 ml of

peptone broth, and the extent of inactivation in agiven time interval was determined by titeringthe phage surviving the treatment by the usualassay procedure. The percentage of survivors orthe reduction in titer after the treatment was cal-culated by comparison with an untreated control.

RESULTS

Serology. An antiserum was first producedagainst W-2a phage and this serum was thentested against all the phages of the collection toreveal serological cross-reactions with otherviruses of the series. The serum against W-2aphage neutralized W-1A, B, and C-131 in additionto the homologous antigen, W-2a. The fourphages, thus related, were therefore placed intoone group which was designated group A. Subse-quently, antisera were produced for the threephages which remained unaffected by W-2aantiserum, i.e., phages W-1, W-3 and W-5. Anti-serum against phage W-1 neutralized W-5 aswell as the homologous phage W-1. The sera

0 30 60 90 120TIME (MIN)

Figure 1. Neutralization curves for group Aphages: per cent survivors determined by reactionof phage at 107 concentration per ml with a 1:1000dilution of W-2a antiserum.

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ALEXANDER AND McCOY

to (I) W-I Serum

0 a0

0~~~~~~

0

V..'

s~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

O0 90 P0O

(2)W-5 Serum

o10.0----!-~~ Wffo

0 40 90 o0TIME (MIN)

Figure B. (1) Neutralization of group B phages upon exposure to W-1 antiserum: W-1 phage(107 particles/ml) was exposed to 1:1000 dilution of the serum, whereas W-5 phage at the same con-centration was reacted with a dilution of 1:100 of W-1 serum. (2) Neutralization of W-5 phage at107 concentration by its homologous antiserum diluted 1:1000.

against W-3 and W-5, however, reacted only withtheir homologous phages W-3 and W-5, respec-tively. Although W-1 serum gave only partialneutralization of W-5 phage (a maxmum of 86per cent was obtained at 1:2 concentration of W-1antiserum), the W-1 and W-5 phages were tenta-tively grouped together and group B was formed.The remaining phage, W-3, was retained in a

group by itself and termed group C. The extentand rate of neutralization of group A phages withW-2a antiserum are shown in figure 1 where thelog of the percentage of free phage after exposureto antiserum is plotted against time. The heterol-ogous phages are neutralized more slowly and to alesser extent by W-2a serum than the homologousW-2a phage, a phenomenon also reported forother phage systems by Burnet (1933a) and byFriedman and Cowles (1953). These curves thusshow that the phages are related but serologicallydistinct from each other. Neutralization of groupB phages, W-1 and W-5, by W-1 antiserum isseen in figure 2 (1) and in figure 2 (2) is shownW-5 phage, reacted with its homologous W-5antiserum. It is interesting to note in comparingthe two curves obtained by subjecting W-5phage to W-1 and W-5 sera, that the shape of thecurves is simiar in that there is an initial dropfoliowed by a long period of little further reaction.The curve for phage W-3 of group C, subjected toits homologous serum, is seen in figure 3.

TIME (MIN)

Figure 3. Neutralization of group C phage by itshomologous antiserum; W-3 serum at 1:1000 dilu-tion was reacted against 10' particles/ml of W-3phage.

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The neutralizing power of an antiserum againsta given phage can also be expressed quantita-tively in terms of a velocity constant k, whichrepresents the fractional rate of neutralization ofthe phage per minute and is a property specificfor the antiphage serum (Hershey et al., 1943).In order to compare different sera it is necessaryto calculate the k value at a reference per cent ofneutralization, which in the present work was setat 99 per cent with a phage concentration of 107particles per ml. The values for the S. griseusphages are found in table 1 and emphasize thedifferences among the various phages. It is thusseen that the S. griseus phage-antiphage reactionsreveal several serological groups and also differ-ences among phages within a group.

One-step growth experiments. The method ofEllis and Delbriick (1939) was used in followingthe growth characteristics of the S. griseus phagesfor the purpose of determining the latent periodsand burst sizes. The experiments were run at30 C, since that is the optimum temperature forgrowth of the host. Grouping of the phages bytheir latent periods (table 2) reveals the con-stancy of the character, as was reported also forthe E. coli phages by Adams (1952). In the S.griseus system it follows closely the serologicalgrouping, with general uniformity within thegroups and considerable difference in time be-tween groups.The latent periods concerned also lend support

to the formation of group B where W-1 and W-5are placed together despite the fact of only partialserological relatedness. Since homologous hostswere used for the one-step growth experiments,several strains of S. griseus were involved. How-ever, it appears that the host is not an influencingfactor on the latent period because W-2a phageon host strain 1947 has a reproducible latentperiod of 90 min whereas phages W-1A and B onthe same host strain have a 120 min period.Similarly phages W-1 and W-3 which also havethe same host require even longer latent periods,160 and 180 min respectively. It will be notedthat these latent periods are exceptionally longcompared with the times reported for mostphage/host systems.Because of the mycelial nature of the host cells,

the burst size characteristic of the S. griuphages might be expected to be more variablethan for other phage systems (Adams, 1952;Friedman and Cowles, 1953; and Luria, 1945).

TABLE 1Classification of the Streptomyces griseus phages by

k values of their neutralizing antiseraSerological Phage Neutralizing k Value/Min atGroup age Serum 99% Neutr.

A W-2a W-2a 153W-1A W-2a 28B W-2a 12C-131 W-2a 4

Serum

W-1 W-5

B W-1 W-1 5 0W-5 W-5 <1* 16

C W-3 W-3 32

* Only 86 per cent neutralization was obtainedwhereas 99 per cent is necessary for calculatingthe k value.

TABLE 2Latent periods and burst sizes of

griseus phagesthe Streptomyces

Phage Latent Period Bunt Size

(mm)Group AW-2a .......... 90 226W-1A.......... 120 340B ........... 120 274C-131*......... 120 118

Group BW-1 ........... 160 168W-5........... 160 125

Group CW-3........... 180 108

Adsorption time was 30 min; the phages wereassayed on their homologous hosts.

* 0.025 m CaCl2 was added; C-131 has particu-larly high Ca++ requirement.

With 15 hr cells of strain 1947 and phage W-2a,Chang (1953) found a range of 300 to more than1000 particles per cell, depending upon conditions,but it was recognized that the apparent numberswere greatly affected by the size of mycelialfragments, counted as cell units. With the 5-6 hrgerminated spores, used in the present study,there was opportunity to determine the burstsize more precisely, which was done with resultsgiven in table 2. The range now appears like thatof other phages reported.

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A comment on the Ca+ requirement of the S.griseus phages is perhaps in order in connectionwith the footnote of table 2. The requirement wasrecognized by Perlman, Langlykke and Roth-berg (1951), and Perlman and Langlykke (1953).For the particular phages in the present studythe optimum for adsorption was determined to be5 X 10- M (Van Astyne, Otto, and McCoy,1955) and this amount is amply provided by thebasal medium here used. However, phage C-131has a particularly high Ca+ requirement (hencethe added 0.025 M CaCl2 in these experiments).When this is added the latent period for phage C-131 conforms to that of the rest of its group, butthe burst size is exceptionally low (table 2).

Morphology. For suitable preparations for elec-tronmicrograph study of morphology the phageswere concentrated and purified by differentialcentrifugation. The preparations were first runat maximum speed in a Sorvall centrifuge for 30min to remove cell debris; then the phage par-ticles were spun down in a Spinco ultracentrifugeat 20,000 rpm in a No. 40 rotor for 1.5 hr. Thesupernatant was discarded and the residue resus-pended in distilled water. After another hour at15,000 rpm, the phage sediment was collectedfrom several tubes and suspended in a total of 2ml of distilled water. The electronmicrographswill appear in a later publication, but the dimen-sions of the particles are pertinent here as addinga valuable characteristic to the classificationscheme. The sizes of the phages are strikinglysimilar for members of group A (table 3). PhagesW-1 and W-5, although comprising group B by

TABLE 3Morphology of the Streptomyces griseus phage8

Particle Size Plaque TypePhage

Head Tail Size Shape

(Mr) (NW) mm

Group AW-1A.. 85 x 75 150 x 15 1.6 CircularW-2a.. 85 x 80 150 x 15 1.4 CircularB. 85 x 80 150 x 15 1.8 IrregularC-131.. 80 x 65 145 x 15 1.4 Circular

Group BW-1... 100 x 100 250 x 25 0.4 IrregularW-5... 70X 55 120x 10 3.6 Halo

Group CW-3... 110 x 85 165 x 15 0.8 Circular

* Indicated measurements represent length xwidth.

TABLE 4Citrate inhibition of multiplication of

Streptomyces griseus phagesReduction in Log Phage Titer from 1 X 107

Phage Per cent citrate added to msay agar

1.00 0.1 0.01 0.001

Group AW-1A... 7.0 1.6 1.3 1.1W-2a. .. 7.0 1.3 0.9 0.8B. 7.0 1.7 1.2 0.7C-131 .. 7.0 1.8 1.3 1.1

Group BW-1 7.0 2.0 1.7 0.8W-5 7.0 1.5 0.4 0.3

Group CW-3 7.0 3.0 1.2 0.8

* Complete inhibition.

other criteria, are very dissimilar with respect toboth particle size and plaque size. Group C withits single phage, W-3, presents distinctive sizes ofits particles and plaques. As can be seen from thetable the usual inverse relationship between par-ticle size and plaque size does hold in general forthese S. griseus phages.InhiWion of phage groth by citrate. The use of

citrate as an inhibitor of phage action was intro-duced by Burnet and Lush (1935) in their classfi-cation of staphylococcal phages. The role ofcitrate is that of a chelating agent for Ca, in themedium, thereby rendering this compound un-available for phage multiplication (Lark andAdams, 1953). This test, therefore, should providean additional differentiating property if thephages concerned differ in Ca+ requirement. Inthe present study it was known that all use theCa+ as co-factor but that at least C-131 re-quired a very high level. A study was thereforemade by adding citrate in varying amounts to theassay medium, and inhibition was recorded interms of the reduction in log phage titer. Thismethod of assay was also employed by Friedmanand Cowles (1953) as a convenient means ofexpressing the extent of inhibition obtained for agiven phage. As seen in table 4, the data cover therange between complete inhibition and measur-able multiplication of phage. For the series as awhole, there appears no drastic difference be-tween the phages; all are inhibited by 1.0 per centcitrate, while measurable survival is obtained at0.1 per cent or less of the chelating agent. Phage

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C-131 does not stand out from the others underconditions of this test; in fact, phage W-3 appearsto be the most citrate sensitive.

Effect of pH on the stability of the phages. Animportant factor affecting stability of phages isthe pH of the suspending medium (Delbruck,1950). The range of the greatest stability for the"T" viruses is reported to be pH 6-8 (Kerbyet al., 1949), and for the "M" phages it is some-what wider, pH 5-10, but within each system, thephages may vary from one another in sensitivity(Friedman and Cowles, 1953).The pH optima for the S. griuec8 phages were

tested with cell-free phage suspensions in 0.1 percent peptone water, subjected to a range of hy-drogen ion concentrations for 1 hr, after whichthe remaining viable phage was plated. As awhole, group A phages are more sensitive to lowpH and more resistant to high pH than are theother phages (table 5). The pH of culture lysatesis generally between 7 and 8 and these valueshave therefore been used for the satisfactorystorage of phage for at least 2 years (Chang,1953).

Thermwl inactivation of the phages. The rate atwhich viral protein is denatured at high tempera-tures is another physical inactivation test whichmay be useful in identification of a phage.Pollard and Reaume (1951) reported the rates ofheat destruction of E. coli phages to be differentfor the four serological groups. Thermal inactiva-tion rates were also determined for classificationof the B. megaterium phages (Friedman andCowles, 1953) and of S. Iactis phages (Wilkowskeet al., 1954b). The S. gri8eus phages were tested

TABLE 5Stability of the Streptomyces griseus phages topH: percentage of survivor8 following 1 hour

exposure to the range pH 4 to 9

Free Phage: Per Cent Survival pH Sta-after Treatment at pH: bility

Phage _ Range4 5 6 7 8 9 Survival)

Group AW-1A.. 2 7 40 100 100 100 7-9W-2a.. 3 9 62 100 100 100 7-9B.. 5 8 76 100 100 100 7-9C-131.. 0 2 78 100 100 62 7-8

Group BW-1.... 18 23 64 100 100 48 7-8W5 .... 5 65 100 100 100 100 6-9

Group CW-3.... 4 74 100 100 100 82 6-8

aor0

at

TIME (MIN)Figure 4. Thermal inactivation of Streptomyces

gri8eus phages at 60 C.

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50

pH1 7.0

x

I0

at

TI ME (MIN)Figure 5. Combined effect of heat and pH on

the rate of inactivation of W-2a bacteriophage:temperature 60 C; pH 6, 7 (control) and 9.

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in cell-free lysates at pH 7.0, subjected to 60 C,since preliminary tests showed that this treatmentrevealed marked differences in rates of inactiva-tion. When the log of the per cent survivors afterexposure to 60 C is plotted against time, a linearrelationship is obtained. A very definite correla-tion between serological grouping and thermalinactivation is seen in figure 4, yet the reaction ofeach phage is distinct. Group A phages are mostresistant to this temperature; groups B and Cmuch less resistant and their rates of inactivationvery similar.The combined effect of heat (60 C) and of

unfavorable pH was examined for one of the moreresistant phages, W-2a. At pH 7.0 thermalinactivation proceeds linearly; at the destructivepH range, especially pH 9, the curve breaks andthen levels off (figure 5). Non-linear curves ofheat inactivation of S. lactis phage are reportedby Wilkowske et al. (1954b) and are attributed tomore resistant particles in the phage population.In the present study it appears that the non-

TABLE 6Survival of W-fa phage after exposure to an un-

favorable pH and a high temperature (60 C)

Percentage of Survivors*pH

Room temperature NC

6 62 1.47 100 22.09 100 6.8

* Exposure to treatment for 1 hr.

linear curves apply only to the destructive com-

bination of heat and unfavorable pH. Similarpatterns applying to two inactivating factorswere obtained for T 5 phage by Lark and Adams(1953) with a combination of high temperature(55 C) and an inhibitory concentration of citrate(0.04 m). The reduction in viable phage at theseinhibitory pH values with and without the appli-cation of heat at 60 C offers a strildng comparison(table 6).

Cross-resitance grouping. Another characteris-tic which may serve in phage classification was

introduced by Burnet and Lush (1935). Themethod of cross-resistance grouping for thedysentery-coli phages resulted in a lytic patterndifferent from that obtained with the sensitivebacterial strains. Colonies of the S. griseus strainsresistant to their respective phages were thereforepicked and the cross tests performed with resultsgiven in table 7. The groups formed are denotedby roman numerals. However, for the classifica-tion of S. griseus phages, this grouping and theserological grouping give little agreement. Ifcross-resistance were used as a criterion forclassification only three phages could be groupedtogether, and each of the others would require a

separate group. Furthermore, the three to begrouped together would be taken from Groups Aand B of the serological systems.

DISCUSSION

In the present classification study, phage prop-

erties were established according to the criteriaproposed by Adams (1953) and used succesully

TABLE 7Original and cross-resistance grouping by lytic pattern

Phage ~Host Strain Lytic Pattern CrossPhe | (Homologous) (Sensitive Hosts) Resistant Strains Lyseds ResistanceGrouping

Group AW-1A. 1947 1947, 1949, C-131 1947/B, C-131/C-131 IW-2a.......... 1947 1947 1947/B, C-131/C-131 IB ............. 1947 1947 C-131/C-131 IVC-131.......... C-131 1947, 1949, C-131 1947/W-2a, 1947/B and 1947/W-1A V

Group BW-1 ........... 1945 1945 1947/B, C-131/C-131 IW-5........... 1949 1947, 1949, C-131 1947/W-2a, 1947/B, C-131/C-131, III

and 1947/W-1AGroup CW-3 ........... 1945 1945, 1947, 1949 and 1947/W-2a, 1945/W-1A, 1947/B, II

C-131 C-131/C-131, and 1947/W-1A

* Sensitive strain/phage to which its cells were made resistant.

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in the identification of phages affecting hosts inthe Eubacteriales. The seven S. griseus phageswere first arranged in three serological groups onthe basis of their cros-reaction patterns withneutralizing antisera according to k values, spe-cific for a given phage-antiphage complex. Theserological groupings were then used as basis onwhich further characterization was superimposed.The morphology of the viruses within the sero-logical groupings generally showed good correla-tion. All S. griseus phages possess a head and atail; however, the size of the head and the lengthof the tail differed greatly for the two phages ofgroup B. Latent periods obtained from one-stepgrowth experiments also supported the groupingby serological means. These latent periods appearto be longer than those of other phage systemsreported to date. Since the latent periods are solong the exact time of lysis is probably not de-termined as closely as that for other phage sys-tems. On the other hand, it affords ample time formanipulation of the host-virus system during theproduction of phage, an advantage in some typesof experiments. Burst sizes for this system fluc-tuate considerably and therefore cannot be con-sidered taxonomically useful; the variability maybe influenced here by the mycelial nature of theS. griseus vegetative cells. When small cell units,such as young germinated spores of about 5 hrage, are used, the burst size falls in the 100-300range.Of the phage inactivation tests used, thermal

inactivation followed serological groupings mostclosely. However, the pH stability range andcalcium ion requirements of this phage series aredistinct for each phage and consequently mayprovide a means of distinguishing between mem-bers within a serological group. Other phagetraits as plaque sizes, host specificity, and cross-resistance patterns are also the attributes ofindividual phages.

Seven viruses active against Streptomyces gri8-eus have been characterized. The claificationscheme formulated is based primarily on theserological grouping of the phages. With the aidof four antisera, three serological groups wereformed. These have been termed A, B, and C andcomprise the following phages: Group A, W-1A,W-2a, B and C-131; Group B, W-1 and W-5; andGroup C, W-3. Other characteristics of the indi-vidual phages determined include: latent periods,burst sizes, morphology, cross-resistance group-

ing, lytic specificity and physical inactivationpatterns.

In addition to providing groupings and dem-onstrating the relationships between the phages,the serological character provides a rapid meansof identification by a comparison of known kvalues with neutralizing antisera. Thus a classi-fication system which had been developed forviruses of true bacteria can be extended to thetaxonomic relationships among viruses active onactinomycetes.

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